Download ORS 2017 Annual Meeting Poster No.1800

Cell-Seeded Adhesive Biomaterial for Repair of Annulus Fibrosus Defects in Intervertebral Discs
Michelle A. Cruz1, Warren W. Hom1, Robert Merrill1, Olivia M. Torre1, Huizi A. Lin2, Philip Nasser1, Andrew C. Hecht1, Svenja Illien-Junger1, James C.
Iatridis1*
1
Leni & Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY United States
2
Biomedical Engineering Department, The City College of New York, New York, NY, United States
INTRODUCTION: Intervertebral disc (IVD) degeneration with herniation is a primary cause for neck/arm and back/leg pain1. After conservative care has
failed, microdiscectomy surgery is the most common treatment to remove herniated nucleus pulposus and annulus fibrosus (AF) fragments.
Microdiscectomy does not repair the AF defect and reherniation and recurrent pain can occur at the operated level 2. Cell therapies for IVD repair show
promise in early clinical trials with capacity to increase cellularity, modulate inflammation, and promote anabolism3. Important design objectives for AF
repair are therefore to deliver cells in a carrier that prevents leakage and unwanted off-target effects of cells4, while also providing biomechanical benefits of
sealing AF defects with little herniation risk. This 3-part study evaluated cell-seeded Genipin-crosslinked Fibrin (FibGen) to develop a cell-seeded adhesive
for repair of AF defects using hydrogel mechanics tests, in situ failure biomechanical tests, and cell culture screening studies. The goal of this study was to
determine optimal fibrin and genipin concentrations that meet mechanical design requirements for AF repair while also promoting cell survival and
extracellular matrix production.
METHODS: Final concentrations of fibrin were 140, 70, or 35 mg/mL and final concentrations of genipin were 6, 2, or 1 mg/mL to create 9 FibGen
formulations (i.e., FxGy where x & y are respective concentrations). All FibGen formulations had final thrombin concentration of 28 U/mL. Part 1:
Hydrogel mechanical testing (n=4-8): Young’s modulus and complex shear modulus were determined by unconfined compression tests from 0-15% strain
(0.2% strain/sec) and by frequency sweep tests in pure shear, respectively (Figure 1A) for 9 FibGen formulations. Part 2: In situ failure biomechanics tests
used bovine caudal motion segments (n=5-9) that were injured with a 4 mm posterolateral biopsy punch defect and repaired with 4 selected FibGen
formulations. Motion segments were compressed to failure (2 mm/min) under a 5° posterolateral flexion (Figure 2A). Part 3: Cell culture screening tests
evaluated 4 FibGen formulations for 7 days. Bovine AF cells were mixed in fibrinogen prior to gelation at a concentration of 20 x 106 cells/mL. Gels were
cultured in hypoxia (5% O2) in low glucose DMEM (10% FBS). Percent cell viability (n=6-8) was assessed by confocal microscopy (Calcein AM and
DAPI). DNA content was quantified (Quantifluor, Promega, Wisconsin, USA) using papain-digested FibGen (n=4). Gene expression of anabolic AF
phenotype markers including Aggrecan and collagens 1, 5 and 12 6,7 were analyzed by TaqMan qPCR (n=4-6). RNA was isolated with Trizol (Life
Technologies, New York USA), cDNA was synthesized (Superscript Vilo cDNA synthesis Kit, Invitrogen), and mRNA at day 7 was quantified relative to
day 1. One-way (formulation) and two-way (formulation and time) ANOVA determined statistical differences using p<0.05 as significant.
RESULTS: Mechanical characterization of FibGen formulations determined that both fibrin and genipin concentration as well as the interaction between
them significantly affected Young’s compressive and complex shear moduli (Figure 1B, C). During in situ failure testing, all repair groups and injured
groups were significantly different from intact motion segments. F140G6 had significantly greater failure strength and subsidence to failure than injured,
although the mean for F140G1 was also greater than Injured and had a trend towards significance (p<0.15) (Figure 2B, C). Cell culture screening tests
showed all formulations had high cell viability, which increased with lower fibrin and genipin concentrations. F35G1 had significantly higher viability
(95%) than F140G6 (85%; Figure 3A). DNA content measurements demonstrated similar effects with significantly greater day 1 DNA content at lower
concentrations, indicating that loss of viability likely occurred with seeding (Figure 3B). F35G1 also had a non-significant decrease in DNA with time.
F70G1 and F35G1 gels had significantly increased gene expression of AF phenotypic markers including Aggrecan, Col 1, Col 5 and Col 12 compared to
F140G6 and F140G1 (Figure 3C).
DISCUSSION: Few adhesive biomaterials exist and this study developed a cell-seeded adhesive biomaterial for AF repair. The original 9 FibGen
formulations screened for mechanical performance were reduced to 4 formulations for continued assessments because they approximated human AF values
or were expected to have improved biological performance. In situ failure tests identified F140G6 to have the greatest failure strength, but 3 formulations
had failure strength values similar to or exceeding high ranges of physiological loading. Although, no FibGen formulations could restore failure strength to
Intact levels. F35G1 and F70G1 formulations had the best cell culture screening performance with significantly greater cell viability and AF gene expression
values than other formulations. Overall, F70G1 appeared to be the formulation that best balanced mechanical and biological performance which provided a
supportive environment for cell viability and matrix production while nearly matching AF tissue properties and having relatively lower failure. Ongoing
studies will measure the long-term mechanical and biological performance of cell-seeded FibGen gels in culture with the eventual goal of advancing towards
organ culture and eventual in vivo assessments.
SIGNIFICANCE: Development of injectable AF repair and IVD tissue regeneration techniques has the potential to improve current treatments for IVD
herniation. This study developed an injectable, cell-seeded adhesive biomaterial with formulations optimized to seal AF defects, reduce reherniation risk,
and promote AF matrix production.
REFERENCES: 1Adams & Dolan J Anat 2012; 2Carragee + Spine 2006 ; 3Sakai & Andersson Nat Rev Rheumatol 2015; 4Vadala + J Tissue Eng Regen
Med 2012 5Likhitpanichkul + Eur Cells Mater 2014; 6van den Akker + PLoS one 2016; 7Clouet + Rheum. 2009.
ACKNOWLEDGEMENTS:
We gratefully acknowledge Dr. Steven Nicoll for important discussion and technical contributions. Funded by NIH grant R01AR057397.
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Figure 1: Mechanical characterization of FibGen formulations with varying fibrin
and genipin concentrations. F140G6, F140G1, F70G1, and F35G1 were selected
for advanced screening. Horizontal grey bars represent range of human AF values.
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B.
A.
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Figure 2: In situ AF repair of bovine IVDs with F140G6 improving failure strength
and subsidence to failure compared to injured. Horizontal grey bar represents range
of human IVD values. (* = p<0.05 compared to intact)
Figure 3: Cells seeded in gels with lower fibrin and genipin
concentrations had higher viability and gene expression suggestive
of an AF phenotype.
ORS 2017 Annual Meeting Poster No.1800